Hours Of Maturation Research Articles

Effect of Numerous Amino Acids During in vitro Fertilization of Ovums and Epididymal Sperm in Iraqi Awassi Sheep

In present experiment, the effect of three amino acids, methionine, L-arginine, and D-alanine, on the process of producing embryos in the laboratory, was studied. A 100 mature ovaries of Awassi sheep slaughtered in the Al-Saadoun slaughterhouse in Nineveh Governorate/Iraq were used. The samples were transported using a refrigerated box after washing with physiological saline and antibiotics. The insemination took place in the IVF laboratory of Soran Hospital in Erbil Governorate from 1/9/2022 to 1/5/2023. An eighty mature eggs with very good characteristics were collected and divided into four groups (20 eggs per group) then specified amino acid was added to each group, three groups (amino acid per group) and the fourth group was left as a control group without addition. The ripening and inoculation process was carried out according to the protocol of the laboratory in which the experiment was conducted. Results of this experiment showed that the arginine group (0.4 Mm) recorded the highest fertilization rate obtained (87.5%) based on the evaluation of embryos when kept in a carbon dioxide incubator (CO2 incubator) after 120 hours of maturation and fertilization, followed by Methionine group (0.2 Mm), control group and finally alanine group (15 Mm), where the results were (85%), (66.6%), (0.0%) respectively depended up on embryo evaluation (Grade A embryo). It is concluded from the current study that the arginine was the best of the three amino acids in stimulating divisions of the egg and improving the percentage of embryos produced in the laboratory after incubation for 120 hours.

Does using microfluidic sperm sorting chips in bovine IVEP affect blastocyst development?

This study aimed to determine the effect of microfluidic sperm sorting chip on embryo development and quality in the sperm treatment step in in vitro embryo production in cattle. Only A-quality oocytes obtained from the ovaries of Holstein cattle were included in the study. These oocytes were first placed in in vitro maturation medium, and matured oocytes were randomly divided into two groups at the 24th hour of maturation. Oocytes in the first group with the Microfluidic Sperm Sorting Chip (MFSC, n = 154) were put into a fertilization medium with spermatozoa prepared with microfluidic sperm sorting device. Oocytes in the second group (Con, n = 169) were fertilized with spermatozoa prepared by the routine sperm treatment step of the commercial company. The rate of cleavage (85.71% vs. 76.33%, respectively) and of reaching the blastocyst (44.15% vs. 32.54%, respectively) in the MFSC group were higher than the control group. In addition, it was determined that the numbers of ICM (45.8 ± 2.04 vs. 39.2 ± 1.85, respectively), TE (122.13 ± 2.19 vs. 115.0 ± 2.61, respectively), TC (167.93 ± 2.89 vs. 154.2 ± 2.62, respectively) increased in the MFSC group compared to the control group. A statistically significant difference was found in the number of cells with apoptosis per embryo (5.14 ± 0.77 vs. 11.91 ± 0.79) and apoptotic index rates (3.06 ± 0.47 vs. 7.72 ± 0.55%) of the MFSC and Con groups. As a result, we concluded that using microfluidic sperm sorting chips during sperm treatment in bovine IVEP increases the rate of reaching the blastocyst, embryo development, and quality and reduces the possibility of apoptosis in developing blastocysts. For this reason, it is also thought that the use of microfluidic sperm sorting devices during sperm treatment in bovine IVEP may be a new alternative in this field.

Effect of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production following ICSI

Melatonin has been shown to have beneficial effects in in vitro oocyte maturation and embryo production through antioxidant activity but also through melatonin receptors on oocytes and cumulus cells as well. The purpose of this study was to investigate the effects of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production with maturation performed in a reduced oxygen atmosphere. Oocytes were recovered via trans-vaginal follicular aspiration and divided randomly into three groups each with 5 replicates. The oocytes were placed in maturation medium containing no added melatonin (Control), 10−9 M melatonin (Group 1), or 10−7 M melatonin (Group 2) in an atmosphere of 5% CO2, 5% O2, and 90% N2. ICSI was performed on metaphase II oocytes after 28-30 hours of maturation. Presumptive zygotes were observed on day 2 post-injection for cleavageand on days 5 through 10 for blastocyst development. Results: No significant difference (p>.01) was determined for maturation to metaphase II, 59/86 (68.6%), 63/88 (71.5%), 66/89 (74.2%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for cleavage 43/59 (72.9%), 43/63 (68.2%), 44/66 (66.7%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for blastocyst formation 19/59 (32.2%), 25/63 (39.6%), 26/66 (39.4%) for Control Group, Group 1, and Group 2 respectively. While there was a slight trend towards an increase in blastocyst production with the addition of melatonin to maturation medium under reduced oxygen concentration, the difference was not statistically significant and further work would be required to evaluate its effect.

EVALUACIÓN DE CUATRO TIEMPOS DE MADURACION IN VITRO EN OVOCITOS DE ALPACA SOBRE LA TASA DE MADURACIÓN Y DESARROLLO EMBRIONARIO

The objective was to evaluate the maturation time to obtain oocytes in Metaphase II (MII) stages and the rate of division and blastocysts after fertilization in alpacas. For this, the ovaries were obtained from benefited animals. Experiment 1: 441 oocytes were distributed in four times (26, 32, 38 and 42 hours) and matured in TCM-199. At the end of the maturation time, the oocytes were fixed in an ethanol solution: acetic acid (3:1) and stained with aceto-orcein (1%). Experiment 2: 959 oocytes were cultured under the same conditions as experiment 1 and fertilized with sperm obtained by artificial vagina and selected by the Swim Up method. At the end of the fertilization time, the presumed zygotes were cultured in SOFaa medium for 7 days. In experiment 1, 39.6 ± 8.0, 49.1 ± 18.6, 45.9 ± 9.1 and 38.2 ± 9.9% of oocytes were obtained in Metaphase-II for 26, 32, 38 and 42 h of maturation, respectively, without statistical difference (p>0.05). In experiment 2, the cleavage rate was 45.7 ± 3.5, 51.3 ± 10.2, 52.8 ± 5.6 and 50.9 ± 3.1 with no statistical difference between treatments (P>0.05) and the blastocyst rate was 20.5 ± 3.4, 30.5 ± 5.8, 21.2 ± 2.5 and 22.8 ± 3.5 for 26, 32, 38 and 42 hours, respectively, with a statistical difference between 32 hours and 26, 38 and 42 hours (p<0.05). The results obtained suggest that 32 hours of maturation are required to obtain 30.5% of blastocysts

In vitro maturation of oocytes from stimulated IVF-ICSI cycles using autologous cumulus cell co-culture: A preliminary study

In stimulated IVF-ICSI cycles, follicles at different stages of maturation can be aspirated during oocyte pickup. Nowadays, only mature oocytes (metaphase 2 stage) are used and immature oocytes (germinal vesicle and metaphase 1 stages), which are judged unfit for fertilization, are non-used at day 0. In our IVF center, the rate of immature oocytes recovered is around 25%. A significant number of this precious resource is therefore non-used every day in IVF laboratories. The objective of our study was to evaluate the competence of our in vitro maturation autologous coculture method on the maturation and developmental potential of immature oocytes obtained from stimulated IVF-ICSI cycles, in order to obtain additional embryos for the couple as a rescue system to increase the changes of cumulative pregnancy. This is a prospective study, carried out in the Reproductive Medicine and Biology Unit of the Amiens-Picardy University Hospital (France). It was included 14 couples, managed in IVF-ICSI in our center, from January to March 2020. Thirty-eight oocytes, identified as immature after cumulus-oocyte complexes (COC) stripping for ICSI, were placed in our in vitro maturation medium with the addition of autologous cumulus cells. Oocytes that had reached the metaphase II stage after a maximum of 36 hours of maturation were microinjected. The fertilization and embryonic development potential of the in vitro matured oocytes were compared to those of 148 in vivo matured "siblings" oocytes from the same oocyte retrieval, and then also compared to those of 127 in vivo matured oocytes from different patients (control group). Maturation rate, fertilization rate, early cleavage rate and developmental activity to blastulation rate. Embryo quality at cleavage and blastocyst stages, blastulation rate, and useful blastulation rate. No significant difference was found in the main and secondary criteria of the study compared to the "siblings" in vivo matured oocytes from the same oocyte retrieval. However, a significant difference was obtained on the rate of early cleavage and useful blastulation when our cohort was compared to mature in vivo oocytes from different patients (control group). This study has shown that after incubation in our in vitro maturation autologous cumulus cell co-culture with cumulus-oocyte cells, immature oocytes recovered during stimulated cycles can give rise to competent oocytes, i.e., capable of being fertilized, of cleaving, and of developing into embryos up to the blastocyst stage. Our study therefore seems to be in the direction of a favorable use of these immature oocytes obtained after stimulated IVF-ICSI cycles. The continuation of this study by including a larger number of oocytes is necessary in order to evaluate the real contribution of this technique in routine.

EVALUATION OF THE MATURATION TIME OF ABATTOIR-SOURCED WATER BUFFALO (Bubalus bubalis) OOCYTES FERTILIZED In vitro

The purpose of this study was to compare the maturation, cellular division, and blastocyst production rates of water buffalo oocytes at 18, 21 and 24 hours of incubation. These oocytes were obtained through follicular puncture, assessed, and matured in BO-IVM medium at 38.5°C, and with a 5% of CO2, at 18 (n=31), 21 (n=34) and 24 hours (n=33). Fertilization was done in a BO-IVF medium at 38.5°C, and with a 5% of CO2, for 18 hours; the maturation rate was assessed a day after fertilization. The embryos were cultured in a BO-IVC medium, at 38.5°C, 5% CO2, 7% O2 and 88% N2, and the cell division rate and number of embryos were assessed at 5 and 7 days after fertilization, respectively. The maturation rate was comparable (P>0.05) among the three groups, while blastocyst production and cell division rates were higher in the group of oocytes matured for 18 hours (P<0.05) than in the 24-hour group. The blastocyst production and cellular division rates in oocytes that were matured for 21 hours were comparable (P>0.05) to the other groups. After 21 hours of maturation, oocytes started to degenerate, which resulted in lower cellular division and blastocyst production rates.

PSXVI-16 The quality of cows’ oocyte derived at different timing regimens of transvaginal follicular puncture

Abstract Recovery of oocytes from live animals through ultrasound-guided transvaginal follicular aspiration (Ovum Pick-Up, OPU) is an essential element of modern livestock development. The aim of our work was to study the effect of two different timing regimens on the quality of oocytes, recovered from cycled Simmental heifers (n = 4). Follicle aspirations were performed once a week (1/w) or twice a week (2/w). The same donor animals were used for each timing regimens. 228 and 224 follicles were aspirated at 2/w and 1/w regimen, respectively, and 280 cumulus-oocyte complexes (COCs) were recovered (140 COCs at each of timing regimen), which corresponds to the recovery rate of 61.4±5.9% and 62.1±6.3%. On average, 4.4 oocytes were received from each donor per session. We observed a significant increase by 1.2 times (P < 0.05) of the proportion of OPU-oocytes of good quality, which were characterized by normal morphology, when performing the procedure in the 2/w compared to 1/w regimen (65.7±4.0% vs. 53.6±3.0% of the total number of recovered oocytes, respectively). The proportion of oocytes at the MII stage of meiosis after 24 hours of maturation was similar in both groups: 73.3±5.6% (2/w regimen) and 74.7±2.4% (1/w regimen). Proportion of oocytes with signs of apoptotic changes was also not significantly differed between the groups: 8.0±3.1 and 9.8±3.3%, respectively. We did not observe the effect of the frequency of OPU sessions on the embryo cleavage rate (on average 63.5%) and the blastocysts development rate (on average 16.7%). Our data will be used to optimize the timing regimen of oocyte retrieval from living Simmental donor heifers. The studies were performed under financial support of the RFBR (project No. 20-316-90003) and the Russian Ministry of Science and Higher Education.

AUTOMATED VITRIFICATION AND WARMING OF OOCYTES MEDIATED BY A NOVEL MICROFLUIDICS DEVICE

The purpose of this study is to develop a microfluidic device to automate the critical process of vitrification and thawing procedures, providing standardization, minimizing inter and intra-center variability, and reducing hands on time. Experimental comparative study of both vitrification and warming of bovine and human oocytes using an automated microfluidics device (Davitri, Overture Life). a microfluidics system has been developed that infuses the solutions required in a central well, where the oocytes are placed, and withdraws the spent solutions. The system and the protocols were first tried in bovine oocytes, which are of similar size as human, and later on human donated oocytes. After 24 hours of maturation the cumulus oocyte complex (COCs) were denuded manually and vitrified using the palm-size Davitri device. The scripts controlling the process are based on the Kitazato protocol. Once the procedure was validated with bovine, frozen human oocytes from donors were warmed and vitrified automatically and fresh human oocytes were vitrified and warmed automatically. 100 fresh bovine oocytes were vitrified automatically and warmed manually, 5 of them were lost during the procedure and 84 oocytes survive after culture (88%), compared to 80% for a control group. Also 50 fresh bovine oocytes were vitrified manually and warmed with the microfluidics device, and 48 of them survived (96%). Donated frozen human oocytes (N=19) were warmed automatically, and 12 survived (63.15%) compared to 80% expected (1,2). Those 12 were re-vitrified automatically and 71% survived thawing compared to 70% expected after re-vitrification (3). After these promising results 4 fresh human mature oocytes from an egg donor were automatically vitrified and manually warmed of which 100% survived. Also, 13 fresh immature oocytes were automatically vitrified and manually warmed of which 11/13 (86.6%) survived. The automation of vitrification and thawing of eggs is an improvement in assisted reproduction by allowing constant high survival rates with reduced hands-on time. Here we provide proof of concept that the Davitri microfluidics device is able to vitrify and warm both bovine and human oocytes with high survival rates.

The Measurement of Shrinkage: Influence of Specimen Size

The paper deals with the experimental determination of the development of shrinkage and mass losses during concrete maturation. Three types of concrete mixtures were prepared for the experiment. The shrinkage measurement was designed to measure the relative length changes along the central axis of the test specimens. The main aim of the experiments was the comparison of the shrinkage and mass loss values using specimens of different dimension. One set of each concrete contained specimens with the dimensions of 100×60×1000 mm. The other set contained specimens with the dimensions of 50×50×300 mm. The measurement of shrinkage and mass losses started approx. 1 hour after the fresh concrete was placed into the moulds. The relative length changes were recorded continuously using an inductive sensor during the first 48 hours of maturation. After the 48 hours, the specimens were demoulded and a long-term measurement was performed manually using a mechanical strain gauge. The outcome of the experiment is an analysis of the influence of specimen size on the resulting curve and values of shrinkage and mass losses.

Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus-oocyte complex in vitro in the presence of hemisections of the follicular walls.

This study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus-oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1-10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (-HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

The influence of a microbiological derived protease on the autolysis process in meat of broiler chickens

The article is devoted to the influence of protease of microbiological origin on the autolysis process in broiler chicken meat. Protease made by Aspergillus fungi was chosen as an enzym. According to the experiment plan, 5 samples were made, including a control sample, in which autolytic processes were performed without the addition of an enzyme. The experimental group (samples I–IV) included samples with 30, 50, 70 and 90 mg of the added enzyme solution, respectively, per 100 g of basic meat raw material. After introduction of the enzyme and parallel fixation of the studied control parameters before maturation, the samples were stored at 4–6 °C for 2 days. The selected physicochemical parameters were studied in all samples after 1 and 2 days of storage. Among the parameters studied, the following were selected: pH, water-holding capacity (WHC), water binding capacity (WBC), fat binding capacity (FBC), moisture content, and optical density by measuring of soluble and water-soluble proteins volume. Examenated transition of proteins from salt- to water-soluble experimental group also as their overall solubility is noticeably higher than in the control sample. Among the functional and technological characteristics, these samples have shown a high pH and WHC levels. The WBC, FBC and total water content show a slight increase. The values of the studied parameters among the samples I–IV have not shown a noticeable difference. For research minced meat at 48 hours of maturation compared with the control, the increase of WHC was accordingly from 16.4 to 18.4%, the value of the university increased by 9.1–19.1%, and FBC by 1.1–5.9%. At the same time, the change in the proportion of salt and algae protein substances of minced meat according to the optical density for 48 hours of maturation was equalized with the control and the variation of this index fluctuated within 1–2 units. A literature and studies sources review have shown the effectiveness of microbial-derived protease enhancement under the action of protease ASP on the functional-technological indices of white meat of broiler chickens. It has been determined that when injecting into broth from chicken broiler meat, ASP proteases in an amount of 30 to 90 mg per 100 g of raw materials, an increase in the thermal stability indices for FBC by 1.1–5.9%, in higher education institutions by 9.1–19.1%, which makes it possible to effectively model the increase in the functional and technological characteristics of minced meat from the amount of enzyme addition and maturation time.

New data concerning oocyte maturationand embryo production in vitro

It is reviewed last science data concerning oocyte maturation and pig embryo production in vitro. The aim of the work is to present new data concerning oocyte maturation and embryo production in vitro. Efficiency of different assistant reproductive methods can be improved by treatment of gametes and embryos with sublethal hydrostatic pressure. COCs denudation is better to perform 44 hours from the initiation of maturation procedure. Last 11 hours of maturation is better to perform in medium with decreased quantity of NaCl. Lipid content in oocyte significantly correlates with number of granulose cells per follicle. Larger size pre- and post pubertal oocytes are characterized by greater ability to development. Supplementation of culture medium with glycine leads to increase number of blastocyst cells. Addition of fibroblast growth factor to COCs culture medium benefits its development and blastocyst formation. COCs maturation normalization decreases polyspermy. Bringing endothelial growth factor in COCs medium culture increases rate of blastocyst formation. Different cells processes require different quantity of calcium ion oscillations. Temperature of liquid of preovulatory follicle can be indicator for oocyte quality. 14-days culture of COCs, recovered from early antral follicles, on polyacrylamide gel, results in achievement of larger size oocytes. It is improved method for optical observation of fertilization. It is worked out morula - blastocyst transportation for long distances. Introduction of Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel. Conclusion: last literature data show that row of different means of improvement for oocyte maturation and obtaining embryo in vitro is worked out.

Análisis Del Efecto De La Adición De Fibra Cítrica Del Bagazo De La Naranja En Las Propiedades Nutrimentales Y Sensoriales De Un Embutido Y Determinación De La Calidad Microbiológica

The orange bagasse (Citrus sinensis) is a residue with a significant content of citrus fiber (FC); it is used as a functional ingredient in the development of new products. We consider it essential to know the effect of the nutritional and sensorial properties of FC. The objective of this research was to evaluate the effect of powdered the orange bagasse on the nutritional and sensorial properties of pork and beef sausage. The sausage was prepared by mixing the proportions of minced pork and beef with the corresponding FC, adding spices, orange juice and a solution of natural dye Bixa Orellana, after 24 hours of maturation was stuffed into cellulose casings. A randomized design with three repetitions was used. The treatments were formulations with 0, 5, 10 and 15% of FC respectively, named treatments E0, E5, E10 and E15. The chemical variables evaluated were: moisture content, ash, fat, protein (analyzed by the AOAC method, 1990), and total dietary fiber (by the methodology described by Asp et al., 1983 and Prosky et al, 1988); the microbiological variables: aerobic mesophiles, total and fecal coliforms, Salmonella and Staphylococcus aureus, detected and quantified with the methods of the corresponding Official Mexican Standards. The sensory variable was the level of liking which was evaluated in 128 consumers, and a commercial sausage called "EC" was used. As a comparison product. In E5, E10 and E15 the fat levels decreased (p &lt;0.05). The ash increased as a function of the addition of FC. Regarding protein, no differences were observed between E0 and E5. In the treatments, E10 and E 15 the protein decreased significantly. Total dietary fiber at E5, E10, and E15 ranged from 4.38 to 10.50%. The consumer test favored E0, E5, and EC. We conclude that the orange bagasse exerts a positive effect by decreasing fat and increasing total dietary fiber. Aleson-Carbonell et al. (2003) report similar behavior. It is concluded that it is feasible to add FC no higher than 5% as an alternative to enrich sausages.

Evaluación sensorial de embutido tipo chorizo a base de carne de conejo

Rabbit meat is a viable option to meet the qualitative and nutritional demands of the current consumers, due to its easy digestion, high protein content and unsaturated fatty acids, low-fat concentration. Despite its nutritional characteristics, this meat is not widely accepted in Mexico, so it is necessary to develop products that favor its acceptance. In the present study, two experiments were carried out using sensory tests to evaluate the acceptance of a sausage type made from rabbit meat. Experiment one consisted of a sensorial test of the sausage with 24 hours of rest after its elaboration, through a hedonic scale with values from 1 to 10 where 1 corresponded to the lowest score and 10 to the highest. Experiment two was developed by a sensorial test for a sausage ripened at 48, 120, 216 and 312 hours, with the hedonic scale established for experiment one. The evaluated aspects of the product were: color, smell, flavor, texture and overall, the results were analyzed by means of descriptive statistics and analysis of variance. In conclusion, the sausage was accepted by the participants in both experiments. In experiment two, the highest level of acceptance corresponded to 216 hours of maturation (P <0.05). Keywords: Maturation, Oryctolagus cuniculus, sensory evaluation.

The effect of salivary pH on diametral tensile strength of resin modified glass ionomer cement coated with coating agent

The aim of this study was to evaluate the effect of artificial saliva with different acidities on the diametral tensile strength of Resin Modified Glass Ionomer Cement (RMGIC) coated with varnish and nanofilled coating agent. The specimens coated with coating agents were immersed in artificial saliva with pH of 4.5, 5.5, and 7 for 24 hours in an incubatorat 37°C. The diametral tensile strength of the specimens was tested with Universal Testing Machine. There were no significant differences on the diametral tensile strength of all specimens that were put into groups based on the acidity of the saliva and the type of coating agent (p>0.05). Both varnish and nanofilled coating agent stayed on the RMGIC in the acidic condition that simulated the true condition of oral cavity in people with high caries risk for the 24 hours of maturation.

L-arginine affects the IVM of cattle cumulus-oocyte complexes

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17β-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control −ve). The addition of 5.0-mM L-arg increased (P < 0.05) the percentage of oocytes at the germinal vesicle breakdown stage after 7 hours of cultivation compared with control −ve. All concentrations of L-arg (2.5, 5.0, and 10.0 mM) increased the percentage of oocytes that reached the metaphase I (MI) at 15 hours (P < 0.05) but do not affect the progression from MI to metaphase II (P > 0.05) at 22 hours. All concentrations of L-arg tested increased (P < 0.05) the percentage of cumulus cells with plasma membrane integrity at 22 hours of cultivation. L-arginine did not change (P > 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control −ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control −ve and treatment with 5-mM L-arg progressively decreased (P < 0.05) after the first hour of cultivation; however, COCs treated with 5.0-mM L-arg had higher concentrations of cGMP at 1 hour of cultivation (P < 0.05). The cAMP concentration of COCs supplemented or not with 5.0-mM L-arg progressively increased until 3 hours of cultivation and at, 6 hours, decreased (P < 0.05). The results show, in using this system, that (1) the mechanisms that give the oocyte the ability to restart the meiosis until MI after adding 5.0-mM L-arg do not involve changes in the concentration of nitrate/nitrite, P4, and E2 in the culture medium and (2) L-arg acts on a pathway that involves changing the cGMP concentration but does not involve changing cAMP concentration. More studies are needed to assess whether the observed effects of L-arg during IVM using this system are via NO or not and what the role is in increasing the viability of cumulus cells in the resumption and progression of meiosis until MI.

Effect of perfluorooctane sulfonate on viability, maturation and gap junctional intercellular communication of porcine oocytes in vitro

Perfluorooctane sulfonate (PFOS) is a broadly used man-made surfactant whose long half-life has led to bioaccumulation. This perfluorinated compound is ubiquitous in human body fluids. PFOS concentrations as high as 26μM in plasma have been reported in occupationally exposed populations, and high levels of PFOS in human follicular fluid have been associated with subfertility. However, the effect of PFOS on the maturation of oocytes in mammals has not been reported to date. The aim of this study was to determine the effects of PFOS during oocyte maturation. Results indicate that PFOS inhibits oocyte viability (Lethal Concentration50=32μM) and maturation (inhibition of maturation50=22μM) at physiologically relevant concentrations. In order to evaluate the mechanisms of oocyte maturation inhibition by PFOS, gap junctional intercellular communication (GJIC) between oocytes and granulosa cells was assessed. GJIC between granulosa cells and the oocyte was significantly affected during the first 8h of maturation. However, the inhibitory effect of PFOS on GJIC was not due to an alteration on the expression of connexin genes Cx43, Cx45 and Cx60. These findings suggest that occupationally exposed populations could be at risk, and that PFOS might affect oocyte maturation by interfering the GJIC in the cumulus-oocyte complexes during the first hours of maturation.

196 ELEVATED TEMPERATURE DURING BOVINE OOCYTE MATURATION ALTERS MITOCHONDRIAL ACTIVITY

Exposure of maturing cumulus-oocyte complexes to a physiologically relevant heat stress altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. To determine if perturbations are coincident with changes in mitochondrial activity, generation of reactive oxygen species (ROS), glutathione and ATP content were examined in maturing oocytes experiencing heat stress. In the first study, ROS content was assessed after 6 h at 38.5, 41, or 42°C using 37 µM 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) or 60 µM dihydrofluorescein diacetate for staining cytoplasmic and mitochondrial ROS, respectively. In a second study, cumulus-oocyte complexes were matured for 0, 12 or 24 h at 38.5°C (control) or 41°C for 12 h (heat stress; 38.5°C thereafter). Total glutathione (GSSG + GSH) and reduced glutathione (GSH) and ATP levels were assessed in cumulus denuded-zona free oocytes as per manufacturer (GSH-Glo Glutathione assay, Promega, Madison, WI, USA; 1 to 25 pmol standards) and ATP determination kit (Life Technologies, Carlsbad, CA, USA; 0.078 to 10 pmol standards). Next, ATP content was assessed in control and heat-stressed oocytes matured for 24 h and in embryos resulting after IVF of control or heat-stressed oocytes (i.e. 2-, 4-, and 8-to-16 cell and blastocyst-stage embryos). Data were analysed as a randomised block design with fixed effects of maturation temperature (and hours of maturation where appropriate), blocking on replicate, using PROC MIXED (SAS 9.2, SAS Institute, Cary, NC, USA). Culture at 41°C for the first 6 h in vitro-matured (IVM) reduced levels of cytoplasmic ROS compared to non-stressed controls (1.02 v. 0.84 fluorescent ratio for 38.5 v. 41°C, SEM = 0.16, P &lt; 0.0001), whereas levels after 42°C were similar to controls (0.94). Mitochondrial ROS were higher after 41°C (1.42) than after 38.5 (0.98) or 42°C (0.98, SEM = 0.14, P &lt; 0.0001). Heat stress exposure increased total glutathione content at 12 h (4.4 v. 5.2 pmol for 38.5 v. 41°C) but levels were decreased by 24 h (6.6 v. 5.9 pmol for 38.5 v. 41°C; SEM = 1.26, P = 0.0002). A similar pattern was found for GSH (P = 0.0001); GSSG was similar across treatments (P = 0.50). Levels of ATP increased during maturation (1.10, 1.20, and 1.65 pmol for 38.5°C at 0, 12, and 24 h) and were increased by heat stress at 24 h (1.65 and 2.01 pmol for 38.5 and 41°C; SEM = 0.31, P = 0.001). In embryos, ATP content was higher in 8-to-16 cell embryos derived from heat-stressed oocytes than in those from control oocytes (P = 0.03); levels were similar in blastocyst-stage embryos (P = 0.21) regardless of origin. In conclusion, application of a physiologically relevant heat stress during maturation altered mitochondrial activity in bovine oocytes. Carryover of effects to the early embryo may explain some of the reductions in embryo development experienced by heat-stressed oocytes. This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.

Alterações na morfologia e na viabilidade no desenvolvimento de embriões bovinos fecundados in vitro com sêmen contaminado experimentalmente à Escherichia coli produtora da toxina Shiga stx2

RESUMO:O objetivo deste trabalho foi avaliar, por meio de micros- copia óptica e eletrônica de transmissão, as alterações na morfologia e a viabilidade do desenvolvimento de embriões bovinos fecundados com sêmen contaminado experimentalmente à Escherichia coli produtora da toxina shiga stx2 (STEC). Para tanto, oócitos foram aspirados de ovários de vacas abatidas e selecionados para maturação in vitro. Após 20-24 horas de maturação, os oócitos foram divididos em 2 grupos. Sendo o primeiro grupo o controle (n = 418), fertilizado com sêmen testado e sem nenhum tipo de contaminante e o segundo, o grupo contaminado (n = 415), fertilizado com sêmen exposto a STEC. Cada sêmen foi tra tado pela técnica de gradiente descontínuo de Percoll. Após o período de fecundação, os embriões foram avaliados quanto a sua morfologia e viabilidade, com o auxílio da microscopia óptica e eletrônica. Na ava liação morfológica, os oócitos fecundados com o sêmen contaminado apresentaram retração citoplasmática, falhas na divisão, assimetria de blastômeros, ooplasma granuloso, coloração castanho-escuro, formação de vacúolos, degeneração e rompimento da zona pelúcida. Essas alte rações não foram observadas no grupo controle. A avaliação de todos oócitos incluídos mostrou taxas de clivagem de 70,3 e 52,8%, respec tivamente, para embriões controle e contaminado (p = 0,0001). Após o 5° dia de desenvolvimento embrionário foram observadas 44,7% de mórulas no grupo controle e 22,4% no grupo contaminado, apresen tando diferença significativa (p=0,0001). A presença da STEC interfere na taxa de clivagem dos embriões e também inviabiliza e provoca queda no desenvolvimento embrionário ao estádio de mórula, além de causar alterações morfológicas durante esse desenvolvimento.

INFLUENCE MATURATION OF VEAL ON THE MICROBIOLOGICAL AND PHYSICAL INDICATORS

The aim of the present study was to evaluate microbial and pH changes in veal meat during maturation. Total viable counts, coliform bacteria and pH changes in chilling meat were evaluated after 24 hours, 7 days and 14 days of meat maturation. There were analysed 8 samples of veal meeat. Results of microbiological analysis were compared with Commission regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Total viable counts (TVC) in samples after 24 hours of chilling ranged from 2.02 log CFU.cm-to 4.21 log CFU.cm-2 (1.64 . 104 CFU.cm-2). The average number of TVC after 24 hours of meat maturation was 3.27 log CFU.cm-2, coliform bacteria (CB) after 24 hours were lower than than 1 log cfu.cm-2 in five samples, and the highest number of coliform bacteria was 1.65 log cfu.cm-2, average number of CB was 1.13 log cfu.cm-2. pH values in samples after 24 hours of maturation were in range from 6.6 to 7.0, average pH value was 6.8. TVC in samples after 7 days of chilling ranged from 3.09 log CFU.cm-2 to 4.01 log CFU.cm-2, the average number of TVC after 7 days of storage was 3.39 log CFU.cm-2. CB after 7 days of meat maturation were lower than 1 log CFU cm-2 in three samples, the highest value of CB was 2.07 log CFU cm-2, the average value of CB in samples after 7 days of meat chilling was 1.03 log CFU cm-2. pH values of meat after 7 days of maturation ranged from 5.5 to 6.1. The average pH value of samples after 7 days of storage was 5.73 pH values of meat after 14 days of maturation ranged from 6.0 to 6.4. The average pH value of samples after 14 days of storage was 6.16.